Fatty-acid based particles

ABSTRACT

The present invention is directed toward fatty acid-based particles, and methods of making such particles. The particles can be associated with an additional, therapeutic agent. Also provided herein is a method of forming fatty acid particles, comprising associating a cross-linked, fatty acid-derived biomaterial with a cryogenic liquid; and fragmenting the bio material/cryogenic liquid composition, such that fatty acid particles are formed. The particles can be used for a variety of therapeutic applications.

This application is a divisional application of U.S. patent applicationSer. No. 12/410,243, filed on Mar. 10, 2009, which is now U.S. Pat. No.8,474,854, the entire disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

The health benefits of certain fatty acids are well-documented. Forexample, the omega-3 fatty acids are essential for heart health and canbe effective in lowering LDL cholesterol levels in the body, stoppingthe buildup of fatty deposits (triglycerides) in the arteries, andincreasing the body's HDL cholesterol levels in the blood.

Omega-3 and omega-6 fatty acids are also known as essential fatty acidsbecause they are important for maintaining good health, even though thehuman body cannot produce such fatty acids. As such, omega-3 and omega-6fatty acids must be obtained from external sources, such as fish oil.Omega-3 fatty acids include eicosapentaenoic acid (EPA), docosahexanoicacid (DHA), and alpha-linolenic acid (ALA). EPA and DHA both haveanti-inflammatory effects and wound healing effects within the humanbody.

Fatty acids are reported to have utility as a coating, stand alonematerial, or formulation ingredient in the delivery of an activepharmaceutical ingredient (see, e.g., US Patent Application PublicationNos. 2006-0067983, 20070202149 and 20080207756). Materials andconstructs composed of fatty acids have demonstrated reducedinflammation and improved healing in-vivo. Alternate physical forms offatty acids, such as particles, either with or without an activepharmaceutical ingredient, would provide an additional means ofdelivering fatty acids and/or pharmaceutical ingredients.

SUMMARY OF THE INVENTION

Accordingly, there remains a need for delivery of fatty acids tosubjects, especially for therapeutic applications (e.g.,anti-inflammatory applications). Certain embodiments of the presentinvention provide fatty acid-based particles. The fatty acid-basedparticles can be used in a variety of therapeutic settings, such as thedelivery of health-benefiting omega-3 fatty acids to a target area. Theparticles can be associated with a therapeutic agent, and therefore canbe used to deliver the therapeutic agent in a controlled manner. Theparticles have the advantage of a high surface area; as such, a largequantity of therapeutic agent can be loaded onto the particles fordelivery to a subject. In addition, the therapeutic loaded fattyacid-based particles can be used in conjunction with medical devices(e.g., vascular grafts, hernia mesh, thin films, stents, etc.). Fattyacid-based particles (with or without a therapeutic agent) also can beloaded into fatty acid based liquids (e.g., fish oil) or gels (e.g.,partially cured fish oil) to create a suspension or an emulsion.

Thus, in one aspect, the invention is directed toward a method offorming fatty acid particles comprising: associating a cross-linkedfatty acid-derived biomaterial with a cryogenic liquid; and fragmentingthe biomaterial/cryogenic liquid composition, such that fatty acidparticles are formed. In one embodiment, the source of the cross-linkedfatty acid-derived biomaterial is a fish oil, e.g., a fish oil that hasbeen heated or exposed to UV-radiation in order to cross link some orall of the fatty acids of the fish oil.

In one embodiment of the method, the step of associating thecross-linked fatty acid-derived biomaterial with a cryogenic liquidcomprises at least one of suspending, submerging, and surrounding thecross-linked fatty acid-derived biomaterial. In another embodiment, thecryogenic liquid comprises liquid nitrogen. The cross-linked fattyacid-derived biomaterial/cryogenic liquid composition can be fragmentedusing one or more of grinding, shearing, shocking, shattering,granulating, pulverizing, shredding, crushing, homogenizing, sonicating,vibrating, and/or milling. The cryogenic liquid can be substantiallyremoved by evaporation, either before fragmentation or after theparticles are formed.

The cross-linked, fatty acid-derived biomaterial can comprise an oilthat may be natural or derived from synthetic sources. The cross-linked,fatty acid-derived biomaterial can comprise a biological oil, such as anoil containing at least one lipid or omega-3 fatty acid, such as a fishoil. The fish oil further can include vitamin E. As described herein,the fish oil is exposed to heating and/or UV irradiation to form across-linked, fatty acid-derived biomaterial (e.g., gel). In oneembodiment, before being associated with a cryogenic liquid, thecross-linked material is in the form of a film. In another embodiment,the film is coarsely ground prior to association with the cryogenicliquid.

When the cross-linked, fatty acid-derived biomaterial is in the form ofa film, a therapeutic agent can be loaded into the film before particleformation, during particle formation, or after particle formation. Instill another embodiment, the film is coated with a therapeuticagent/solvent mixture. The therapeutic agent can be dissolved in asolvent, such as methanol or ethanol, and the therapeutic agent/solventmixture can be applied to the film, e.g., by dipping or spraying.

Once prepared, the fatty acid particles can be soaked in a therapeuticagent dissolved in solvent, such as hexane, isopar, water, ethanol,methanol, proglyme, methylene chloride, acetonitrile, acetone, or MEK,and the solvent can be substantially removed, resulting in fatty acidparticles associated with a therapeutic agent.

The particles, either alone or loaded with a therapeutic agent, can thenbe used for controlled and targeted delivery in vivo. Thus, in variousembodiments, the present methods facilitate increasing the utility of atherapeutic agent, e.g., increased bioavailability, and improvement inrelease profile from a sustained release formulation.

The therapeutic agent used herein can be one or more of an antioxidant,anti-inflammatory agent, anti-coagulant agent, drug to alter lipidmetabolism, anti-proliferative, anti-neoplastic, tissue growthstimulant, functional protein/factor delivery agent, anti-infectiveagent, imaging agent, anesthetic agent, chemotherapeutic agent, tissueabsorption enhancer, anti-adhesion agent, germicide, analgesic,antiseptic, or pharmaceutically acceptable salts, esters, or prodrugsthereof. In particular embodiments, the therapeutic agent is selectedfrom the group consisting of rapamycin, marcaine, Cyclosporine A(referred to herein as “CSA”), ISA 247 (referred to herein as “ISA”) andrifampicin.

In another aspect, provided herein is a method of forming therapeuticfatty acid particles comprising: (a) combining a cross-linked, fattyacid-derived biomaterial (e.g., a cross-linked fish oil) and atherapeutic agent to form a first composition; (b) submerging,surrounding, or suspending the composition in a cryogenic liquid (c)fragmenting the composition; and (d) optionally removing the dispersingmedia. In one embodiment, the dispersing media comprises a solvent thatwill not dissolve the therapeutic agent or the cross-linked, fattyacid-derived biomaterial. In still another embodiment, the solvent ishexane, Isopar, water, ethanol, methanol, Proglyme, methylene chloride,acetonitrile, acetone, MEK, liquid nitrogen, and other solvents that donot fully dissolve the therapeutic agent. In another embodiment, thecross-linked, fatty acid-derived biomaterial is in the form of a film.In another embodiment, the film is coarsely ground prior to associationwith the therapeutic agent.

In one embodiment, the mean particle size of the particles produced bythe methods described herein is in the range of about 1 micron to about50 microns, e.g., 1 micron to about 10 microns. In another embodiment,the particles have a distribution of size of about 1-20 μm (v,0.1),21-40 μm (v,0.5), and 41-150 μm (v,0.9).

In another aspect, the invention provides a medical device, comprising:a medical device structure, and a coating formed on at least a portionof the medical device structure, wherein the coating comprises fattyacid particles of the invention. The medical device structure can be avascular graft, hernia mesh, thin film, or stent.

In another aspect, provided herein are therapeutic fatty acid particlescomprising a fatty acid. In one embodiment, the therapeutic fatty acidparticles comprise a fatty acid and a therapeutic agent. The source ofthe fatty acid can be a fish oil. The particles can be further combinedwith a fish oil to form a therapeutic composition.

In still another aspect, the invention provides a method of making afilm comprising exerting pressure on the fatty acid particles such thata film is formed. Thus, provided herein is a therapeutic film comprisingpressed therapeutic particles, wherein the therapeutic particlescomprise a fatty acid and optionally a therapeutic agent. The film canhave enhanced mechanical properties. The particles used to form the filmcan also comprise a therapeutic agent. As discussed herein, the film canbe used for a number of therapeutic applications. The particles can bepressed into a film using, e.g., a Carver Press.

In still another aspect, the invention provides a method of makingthree-dimensional articles comprising exerting pressure on the fattyacid particles such that a three dimensional article is formed. Thus,provided herein is a three-dimensional article comprising pressedtherapeutic particles, wherein the therapeutic particles comprise afatty acid and optionally a therapeutic agent. The three-dimensionalarticle can be formed from the particles by any applicable method knownin the art, including, but not limited to, molding, casting, orextruding.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other aspects, embodiments, objects, features andadvantages of the invention can be more fully understood from thefollowing description in conjunction with the accompanying drawings. Inthe drawings like reference characters generally refer to like featuresand structural elements throughout the various figures. The drawings arenot necessarily to scale, emphasis instead being placed uponillustrating the principles of the invention.

FIG. 1 is a 20× magnified photograph of fatty-acid particles producedusing the methods described herein.

FIG. 2 is a graphical representation of the elution profile of fattyacid-based particles soaked in a rapamycin/solvent combination accordingto the methods described herein.

FIG. 3 is a graphical representation of the elution profile of across-linked, fatty acid-derived biomaterial film loaded byrapamycin/solvent spray prior to particle formation according to themethods described herein.

FIG. 4 is a graphical representation of the elution profile of fattyacid-based particles soaked in Cyclosporin A/solvent combinationaccording to the methods described herein.

FIG. 5 is a graphical representation of the elution profile of across-linked, fatty acid-derived biomaterial film loaded byMarcaine/solvent spray prior to particle formation according to themethods described herein.

FIG. 6 is a graphical representation of the elution profile of fattyacid-based particles loaded with rapamycin using different methodsaccording to the methods described herein.

FIG. 7 is a plot showing the average dissolution of two 100 mg samplesof the particles described herein in 0.1M PBS.

FIG. 8 is a plot demonstrating the average dissolution curve of 1×3″film samples prior to particle formation.

FIG. 9 is a plot demonstrating the average dissolution of GentamicinSulfate from a film formed of particles described herein.

FIG. 10 is a graphical representation of zone of inhibition results froma Gentamicin Sulfate loaded film formed of fatty acid-based particlesdescribed herein.

FIG. 11 is a plot demonstrating the average dissolution of GentamicinSulfate from a mesh coated with a film formed of fatty acid-basedparticles described herein.

FIG. 12 is a graphical representation of the effectiveness scores from arabbit peridural fibrosis spine model using a particle material formedwith fatty acid-based particles described herein.

FIGS. 13A-13E are various images of medical devices that can be coatedwith the particles of the invention.

DETAILED DESCRIPTION

As discussed below, the particles described herein are biocompatible,meaning they are compatible with living tissue (e.g., they are nottoxic, injurious, or physiologically reactive), and they hydrolyze intonon-inflammatory components that are subsequently bio-absorbed bysurrounding tissue. As such, the cross-linked, fatty acid-derivedmaterials used to form the particles are referred to as “biomaterials.”

In one embodiment, the cross-linked, fatty acid-derived biomaterials arenon-polymeric. In another embodiment, the cross-linked, fattyacid-derived biomaterial is a gel, e.g., a gel formed from fish oil.

Cross-linked, fatty acid-derived biomaterials in particle form can beadvantageous over other forms of material (such as films) due to theincreased surface area of the particles. A cross-linked, fattyacid-derived biomaterial in particle form that is physiologicallyacceptable, bioabsorbable, and does not induce an inflammatory responsecan have a significant advantage in drug delivery over current deliverymethods. In addition, the cross-linked fatty acid derived particle canbe very useful in the delivery of therapeutic agents, e.g.,water-insoluble therapeutic agents. As discussed below, fatty-acid basedparticles can also be used without the addition of therapeutic agentsfor applications such as adhesion minimization in vivo.

Cross-linked, fatty acid-derived biomaterials in particle form loadedwith a therapeutic agent can be used independently to deliver thetherapeutic agent in a controlled manner. In addition, the therapeuticloaded fatty acid-derived particles can be used in conjunction withother medical devices (e.g., vascular grafts, hernia mesh, thin films,stents, etc.). These fatty acid-based particles (with or without atherapeutic agent) can also be loaded into fatty acid-based liquids(e.g., fish oil) or gels (e.g., partially cured fish oil) to create anemulsion or suspension.

In various aspects, embodiments can provide methods for formingparticles from cross-linked, fatty acid-derived biomaterials. In variousembodiments the composition comprises a cross-linked, fatty acid-derivedbiomaterial (e.g., a cross-linked fish oil), a therapeutic agent, or acombination thereof. The method comprises associating the compositionwith a cryogenic liquid, and fragmenting the composition into aplurality of particles of desired size using a variety of methods, andremoving the cryogenic liquid. In various embodiments, a therapeuticagent is loaded into the cross-linked, fatty acid-derived biomaterial.The therapeutic agent can be added before the composition is fragmented,while the composition is being fragmented, or after the composition hasbeen fragmented.

Suitable fragmentation methods include, but are not limited to,grinding, shearing, shocking, shattering, granulating, pulverizing,shredding, crushing, homogenizing, sonicating, vibrating, and/ormilling. Suitable means for fragmenting the cross-linked, fattyacid-derived biomaterial into solid particles include, but are notlimited to, mills, e.g., screening mills and impact mills such as hammermills, and homogenizers, e.g., rotor-stator homogenizers.

Cryogenic Materials for Particle Formation

In various aspects, embodiments can provide methods of producing fattyacid particles. The particles may comprise a cross-linked, fattyacid-derived biomaterial, and optionally a therapeutic agent. Using themethods described herein, cross-linked, fatty acid-derived biomaterialsin the form of particles of various particle size can be produced.

As used herein, the term “particle,” “particle form,” or “fattyacid-based particle” includes solid, partially solid, and gel-likedroplets and microcapsules that incorporate a solid, partially solid,gel-like or liquid cross-linked, fatty acid-derived biomaterial, e.g., across-linked fish oil. Particles provided and employed herein may have anominal diameter as large as 999 nanometers, e.g., about 1 micron toabout 50 microns.

As used herein, the term “particle size” refers to a number mediandiameter or a volume median diameter as determined by conventionalparticle size measuring techniques known to those skilled in the artsuch as, for example, laser diffraction, photon correlationspectroscopy, sedimentation field flow fractionation, diskcentrifugation, electrical sensing zone method, or size classificationsuch as sieving. “Particle size” can also refer to the minimum dimensionof a population of particles. For example, particles that are sizeclassified by sieving can have a minimum dimension that is no greaterthan the size of the holes contained in the sieve.

In various embodiments, the methods described herein may yield particleshaving a mean particle diameter of 1 micron to about 50 microns, morespecifically 1 to 10 microns. In certain embodiments, the methods canyield particles having a mean particle diameter of less than about 1micron, e.g., 500-999 nanometers. The size of the resulting particles isdependent on the processing conditions and individual characteristics ofthe composition being processed.

In various embodiments, the method can include the step of fragmenting acomposition, e.g., a cross-linked, fatty acid-derived biomaterial, atherapeutic agent, or a combination thereof, while associated with acryogenic fluid, e.g., liquid nitrogen, thereby forming the particles ofdesired size. As used herein the term “cryogenic fluid” or “cryogenicliquid” refers to liquefied gases that are maintained in their liquidstate at very low temperatures. Exemplary cryogenic fluids include, butare not limited to, liquid nitrogen, liquid helium, liquid neon, andliquid argon. In various embodiments, the cryogenic liquid used isliquid nitrogen. Alternate cryogens are listed in the table below:

Gas Normal ° C. Helium - 3 −269.9 Helium -4 −268.9 Hydrogen −252.7Deuterium −249.5 Tritium −248.0 Neon −245.9 Nitrogen −195.8 CarbonMonoxide −192.0 Fluorine −187.0 Argon −185.7 Oxygen −183.0 Methane−161.4 Krypton −151.8 Tetrafluromethane −128.0 Ozone −111.9 Xenon −109.1Ethylene −103.8 Boron trifluoride −100.3 Nitrous Oxide −89.5 Ethane−88.3 Hydrogen chloride −85.0 Acetylene −84.0 Fluoroform −84.01,1-Difluoroethylene −83.0 Chlorotrifluoromethane −81.4 Carbon Dioxide−78.5 (sublimes)

The compositions can be associated with a cryogenic liquid in a varietyof ways, including, but not limited to, being suspended, submerged,surrounded, or cooled by a cryogenic liquid. In various embodiments, thecomposition is directly associated with the cryogenic liquid, i.e., thecomposition itself is in contact with the cryogenic fluid. For example,the composition can be suspended in the cryogenic liquid, e.g., liquidnitrogen. The composition can also be indirectly associated with thecryogenic liquid, i.e., the composition is not in contact with cryogenicliquid. For example, the composition can be contained in a vial, and thevial is then suspended, submerged, surrounded, or cooled by a cryogenicliquid.

In various embodiments, the cryogenic liquid is substantially removedafter the fragmentation step. One of ordinary skill in the art willrecognize methods by which to substantially remove the cryogenic liquidfrom the composition. For example, the cryogenic liquid, e.g., liquidnitrogen, can be removed by vacuum evaporation to increase the rate ofevaporation.

Thus, in one embodiment, provided herein is a method of preparing afatty-acid derived particle comprising heating a fatty acid-containingmaterial (e.g., a fish oil) to form a cross-linked, fatty acid-derivedbiomaterial. The resulting material is then contacted with a cryogenicmaterial (e.g., liquid nitrogen), and then fragmented into particleform.

In another embodiment, the cross-linked, fatty acid-derived biomaterialcan be first cured to form a film. The resulting film is then contactedwith a cryogenic material (e.g., liquid nitrogen), and then fragmentedinto particle form. Alternatively, the film can be ground to particleform in the absence of a cryogenic liquid. A therapeutic agent can beassociated with the particles, either by combing the agent with thefatty acid-containing material before or after heating, combining theagent with the film, and/or combining the agent with the resultingparticles. In a non-limiting example, the agent can be dissolved in asolvent (e.g., methanol or ethanol), and sprayed onto or soaked with thematerial of interest.

As used herein, the term “suspension” or “suspended” refers to a mixtureof two substances, one of which is finely divided and dispersed in theother. For example, in various embodiments the composition, e.g., atherapeutic agent alone, a cross-linked, fatty acid-derived biomaterial,or a combination thereof, is dispersed in the cryogenic liquid, e.g.,liquid nitrogen.

The term “emulsion,” as used herein, includes classic oil-in-water orwater in oil dispersions or droplets, as well as other lipid structuresthat can form as a result of hydrophobic forces that drive polarresidues (i.e., long hydrocarbon chains, such as those found in fattyacids) away from water and drive polar head groups toward water, when awater immiscible oily phase is mixed with an aqueous phase. These otherlipid structures include, but are not limited to, unilamellar,paucilamellar, and multilamellar lipid vesicles, micelles, and lamellarphases. Such systems possess a finite stability, generally defined bythe application or relevant reference system, which may be enhanced bythe addition of amphiphilic molecules or viscosity enhancers.Accordingly, the fatty acid-based particles provided herein (with orwithout a therapeutic agent) also can be loaded into fatty acid basedliquids (e.g., fish oil) or gels (e.g., partially cured fish oil) tocreate an emulsion.

As used herein, the term “dispersing media” refers to any substancecapable of dispersing one or more substances within it, withoutdissolving the dispersed substance. Examples of dispersing mediainclude, but are not limited to, hexane, Isopar, water, ethanol,methanol, nMP, Proglyme, methylene chloride, acetonitrile, acetone, MEK,and liquid nitrogen.

Suitable fragmentation methods include, but are not limited to,grinding, shearing, shocking, shattering, granulating, pulverizing,shredding, crushing, homogenizing, sonicating, vibrating, and/ormilling. Suitable means for fragmenting the solid particles include, butare not limited to, mills, e.g., screening mills and impact mills suchas hammer mills, and homogenizers, e.g., rotor-stator homogenizers. Anexample of a suitable mill for fragmenting the material to formparticles is the Silverson L4R Homogenizer (Silverson Machines, Inc.,East Longmeadow, Mass.). In various embodiments, the starting materialsare fragmented into particles by grinding the materials with a mortarand pestle while the materials are suspended in liquid nitrogen.

In various embodiments, the starting materials are fragmented into solidparticles by impacting the starting materials with a rod that ismagnetically actuated. For example, a Spex Certiprep Cryomill (model6750) can be used to fragment solid materials into particles. Thecomposition can be placed in an enclosed vial, and a rod like impactoris enclosed in the vial. The vial is maintained at cryogenictemperatures, and the rod is rapidly oscillated in the vial by means ofmagnets.

The extent to which particle size is formed from the starting materialsis dependent on the selected processing parameters, such as the lengthand number of cycles and the speed of the impactor (impacts/second). Forexample, if a Spex Certiprep Cryomill is used to fragment the startingmaterials into particles, the size of the vial the composition iscontained in, the amount of composition fragmented, and the size of theimpactor will affect the resulting particle size.

In various aspects, a composition comprising a cross-linked, fattyacid-derived biomaterial (e.g., a cross-linked fish oil) is fragmented,thereby resulting in formation of the particles. The cross-linked, fattyacid-derived biomaterial, e.g., the non-polymeric cross-linked fish oil,is bio-absorbable. As utilized herein, the term “bio-absorbable”generally refers to having the property or characteristic of being ableto penetrate a tissue of a patient's body. In certain embodiments,bio-absorption occurs through a lipophilic mechanism. The bio-absorbablesubstance can be soluble in the phospholipid bi-layer of cells of bodytissue. It should be noted that a bio-absorbable substance is differentfrom a biodegradable substance. Biodegradable is generally defined ascapable of being decomposed by biological agents, or capable of beingbroken down by microorganisms or biological processes. Biodegradablesubstances can cause inflammatory response due to either the parentsubstance or those formed during breakdown, and they may or may not beabsorbed by tissues.

Cross-Linked, Fatty Acid-Derived Biomaterial

In various embodiments, the cross-linked, fatty acid-derived biomaterialcan be derived from fatty acid compounds. The fatty acids includeomega-3 fatty acids when the oil utilized to form the cross-linked,fatty acid-derived biomaterial is fish oil or an analog or derivativethereof. Although some curing methods can have detrimental effects on atherapeutic agent combined with an omega-3 fatty acid oil startingmaterial, one characteristic that can remain after certain curing by,e.g., heating and UV irradiation methods, is the non-inflammatoryresponse of tissue when exposed to the cured omega-3 fatty acidmaterial. As such, an oil containing omega-3 fatty acids can be heated,UV irradiated, or both for curing purposes, and still maintain some oreven a majority of the therapeutic effectiveness of the omega-3 fattyacids. In addition, although the therapeutic agent combined with theomega-3 fatty acid and cured with the omega-3 fatty acid can be renderedpartially ineffective, the portion remaining of the therapeutic agentcan maintain pharmacological activity and in some cases be moreeffective than an equivalent quantity of agent delivered with othercoating materials.

As liquid fish oil is heated, autoxidation occurs with the absorption ofoxygen into the fish oil to create hydroperoxides in an amount dependentupon the amount of unsaturated (C═C) sites in the fish oil. However, the(C═C) bonds are not consumed in the initial reaction. Concurrent withthe formation of hydroperoxides is the isomerization of (C═C) doublebonds from cis to trans in addition to double bond conjugation. It hasbeen demonstrated that hydroperoxide formation increases withtemperature. Heating of the fish oil allows for cross-linking betweenthe fish oil unsaturated chains using a combination of peroxide(C—O—O—C), ether (C—O—C), and hydrocarbon (C—C) bridges. The formationof the cross-links results in gelation of the fish oil. The heating canalso result in the isomerization of cis (C═C) bonds into the transconfiguration. The (C═C) bonds can also form C—C cross-linking bridgesin the glyceride hydrocarbon chains using a Diels-Alder Reaction. Inaddition to solidifying the material (e.g., a fish oil fatty acid)through cross-linking, both the hydroperoxide and (C═C) bonds canundergo secondary reactions converting them into lower molecular weightsecondary oxidation byproducts including aldehydes, ketones, alcohols,fatty acids, esters, lactones, ethers, and hydrocarbons.

UV initiated curing (photo-oxygenation) involves the interaction betweena double bond and singlet oxygen produced from ordinary triplet oxygenby light and typically in the presence of a sensitizer such aschlorophyll or methylene blue and results in the formation ofhydroperoxides. The chemical reaction is described in the followinggraphic.

Because the above described reaction is not a radical chain process, thereaction possesses no induction period and is typically unaffected byantioxidants commonly used to inhibit autoxidation. However, thisreaction can be inhibited by single oxygen quenchers such as carotene.This reaction is limited to C═C carbon atoms and results in a conversionfrom cis to trans C═C isomers during curing (as occurs with heatinitiated curing). However, photo-oxygenation using UV is a relativelyquicker reaction than autoxidation from heat curing, in the realm ofabout 1000-1500 times faster. The quicker reaction especially holds truefor methylene interrupted polyunsaturated fatty acids, such as EPA andDHA, which are found in the fish oil based embodiments.

An important aspect of UV curing when compared to heat curing is thatalthough the byproducts obtained by both curing methods are similar,they are not necessarily identical in amount or chemical structure. Onereason for this is due to the ability of photo-oxygenation to createhydroperoxides at more possible C═C sites as shown for linolenate in thebelow graphic.

Photo-oxygenation, such as that which results from UV curing, due to itsenhanced ability to create inner hydroperoxides, also results in theability to form relatively greater amounts of cyclic byproducts, whichalso relates to peroxide cross-linking between fish oil hydrocarbonchains. For example, photo-oxygenation of linolenate results in 6different types of hydroperoxides to be formed where autoxidationresults in only 4. The greater amount of hydroperoxides created usingphoto-oxygenation results in a similar, but slightly different,structure and amount of secondary byproducts to be formed relative toautoxidation from heat curing. Specifically, these byproducts arealdehydes, ketones, alcohols, fatty acids, esters, lactones, ethers, andhydrocarbons.

Accordingly, in various embodiments, the cross-linked, fattyacid-derived biomaterial that comprises the fatty acid-based particlescan be derived from fatty acid compounds, such as those of fish oil,that include a cross-linked structure of triglyceride and fatty acidmolecules in addition to free and bound glycerol, monoglyceride,diglyceride, and triglyceride, fatty acid, anhydride, lactone, aliphaticperoxide, aldehyde, and ketone molecules. Without being bound by theory,it is believed that there are a substantial amount of ester bondsremaining (i.e., “partially cross-linked, non-polymeric”) after curingin addition to peroxide linkages forming the majority of the cross-linksin the material. The cross-linked, fatty acid-derived biomaterialdegrades (e.g., by hydrolysis) into fatty acid, short and long chainalcohol, and glyceride molecules, which are all substantiallynon-inflammatory and likewise can be consumable by cells, such as, e.g.,smooth muscle cells. Thus, the cross-linked, fatty acid-derivedbiomaterial is bio-absorbable and degrades into substantiallynon-inflammatory compounds.

The bio-absorbable nature of the gel component of the cross-linked,fatty acid-derived biomaterial that comprises the fatty acid-basedparticles can result in the cross-linked, fatty acid-derived biomaterialbeing absorbed over time by the cells of the body tissue such thatsubstantially none remains. In various embodiments, there aresubstantially no substances in the cross-linked, fatty acid-derivedbiomaterial, or breakdown products, that induce an inflammatoryresponse. For example, in various embodiments, the cross-linked, fattyacid-derived biomaterial upon break-down does not produce either lacticacid or glycolic acid break-down products in measurable amounts. Thepreferred cross-linked, fatty acid-derived biomaterial is generallycomposed of, or derived from, omega-3 fatty acids bound totriglycerides, potentially also including a mixture of free fatty acidsand vitamin E (alpha-tocopherol). The triglycerides are broken down bylipases (enzymes) which result in free fatty acids that can then betransported across cell membranes. Subsequently, fatty acid metabolismby the cell occurs to metabolize any substances originating with thematerial. The bio-absorbable nature of the fatty acid-based particlesresults in the material being absorbed over time.

An advantage of the cured fish oil is that the curing conditionsutilized (i.e., cure time and temperature) can directly influence theamount of cross-linking density and byproduct formation, which in turneffects the degradation. Thus, by altering the curing conditionsemployed, the dissolution rate of a therapeutic compound of interestcontained in the cross-linked, fatty acid-derived biomaterial can alsobe altered.

In various embodiments, an agent, such as, e.g., a free radicalscavenger, can be added to the starting material to tailor the drugrelease profile of the cross-linked, fatty acid-derived biomaterial thatcomprises the fatty acid-based particles. In various embodiments,antioxidants, e.g., vitamin E, are added to the starting material to,for example, slow down autoxidation in fish oil by reducinghydroperoxide formation, which can result in a decrease in the amount ofcross-linking observed in a cured fish oil material. In addition, otheragents can be used to increase the solubility of a therapeutic agent inthe oil component of the starting material, protect the drug fromdegradation during the curing process, or both. For example vitamin Ecan also be used to increase the solubility of certain drugs in a fishoil starting material, and thereby facilitate tailoring the drug load ofthe eventual cured coating. Thus, varying the amount of Vitamin Epresent in the coating provides an additional mechanism to alter thecross-linking and chemical composition of the cross-linked, fattyacid-derived biomaterial that comprises the fatty acid-based particles.

In various aspects, the cross-linked, fatty acid-derived biomaterialcontains a therapeutic agent. In various embodiments, a therapeuticagent is combined with a fatty acid compound prior to formation of theparticle. The resultant particle has the therapeutic agent interspersedthroughout the particle.

The cross-linked, fatty acid-derived biomaterial is formed from an oilcomponent. The oil component can be either an oil, or an oilcomposition. The oil components can comprise one or more of naturallyoccurring oils, such as fish oil, cod liver oil, cranberry oil, or otheroils having desired characteristics. In various embodiments, the oil,e.g., fish oil, is exposed to processing steps, e.g., heating orUV-radiation, to form a cross-linked, fatty acid-derived biomaterial,e.g., a cross-linked fish oil. The material (e.g., fish oil that hasfully or partially cross-linked fatty acids) can be further exposed toprocessing steps, e.g., heating or UV-radiation, to form a film. Invarious aspects, the film is suspended in liquid nitrogen, andfragmented, thereby forming particles from the film. In various aspects,the film is suspended in liquid nitrogen and fragmented into particles,combined with an oil, e.g., fish oil, and again cryoground into the fishoil thereby producing a substantially thickened formulation.

In various embodiments, a fish oil is used in part because of the highcontent of omega-3 fatty acids, which can provide, e.g., healing supportfor damaged tissue, as discussed herein. The fish oil can also serve asan anti-adhesion agent. The fish oil can also maintain anti-inflammatoryand/or non-inflammatory properties. The present invention is not limitedto formation of the cross-linked, fatty acid-derived biomaterialformulation with fish oil as the naturally occurring oil. However, thefollowing description makes reference to the use of fish oil as oneexample embodiment. Other oils, such as naturally occurring oils orsynthetic oils, can be utilized.

It should be noted that as utilized herein, the term “fatty acid”includes, but is not limited to, omega-3 fatty acid, fish oil, freefatty acid, monoglycerides, di-glycerides, or triglycerides, esters offatty acids, or a combination thereof. The fish oil fatty acid includesone or more of arachidic acid, gadoleic acid, arachidonic acid,eicosapentaenoic acid, docosahexaenoic acid or derivatives, analogs andpharmaceutically acceptable salts, esters, or prodrugs thereof.

As utilized herein, the term “free fatty acid” includes, but is notlimited to, one or more of butyric acid, caproic acid, caprylic acid,capric acid, lauric acid, myristic acid, palmitic acid, palmitoleicacid, stearic acid, oleic acid, vaccenic acid, linoleic acid,alpha-linolenic acid, gamma-linolenic acid, behenic acid, erucic acid,lignoceric acid, methyl and ethyl esters of fatty acids, fatty alcohols,and analogs and pharmaceutically acceptable salts thereof.

With regard to oils, the greater the degree of unsaturation in the fattyacids the lower the melting point of a fat, and the longer thehydrocarbon chain the higher the melting point of the fat. Apolyunsaturated fat, thus, has a lower melting point, and a saturatedfat has a higher melting point. Those fats having a lower melting pointare more often oils at room temperature. Those fats having a highermelting point are more often waxes or solids at room temperature. A fathaving the physical state of a liquid at room temperature is an oil. Ingeneral, polyunsaturated fats are liquid oils at room temperature, andsaturated fats are waxes or solids at room temperature.

Polyunsaturated fats are one of four basic types of fat derived by thebody from food. The other fats include saturated fat, as well asmonounsaturated fat and cholesterol. Polyunsaturated fats can be furthercomposed of omega-3 fatty acids and omega-6 fatty acids. Unsaturatedfatty acids are named according to the position of its first double bondof carbons, those fatty acids having their first double bond at thethird carbon atom from the methyl end of the molecule are referred to asomega-3 fatty acids. Likewise, a first double bond at the sixth carbonatom is called an omega-6 fatty acid. There can be both monounsaturatedand polyunsaturated omega fatty acids.

Omega-3 and omega-6 fatty acids are also known as essential fatty acidsbecause they are important for maintaining good health, despite the factthat the human body cannot make them on its own. As such, omega-3 andomega-6 fatty acids must be obtained from external sources, such asfood. Omega-3 fatty acids can be further characterized as containingeicosapentaenoic acid (EPA), docosahexanoic acid (DHA), andalpha-linolenic acid (ALA). EPA and DHA both have anti-inflammatoryeffects and wound healing effects within the human body.

Cryogenic Fragmentation

The methods provided herein include the step of fragmenting compositionswhile associated with a cryogenic fluid, e.g., liquid nitrogen, therebyforming particles. The composition is cooled to cryogenic temperature toensure that the materials are brittle enough to grind into micron andsubmicron particle sizes. The cryogenic conditions preserve the physicaland chemical characteristics of the ground products from thermal damage.Cryogrinding can achieve a more homogeneous particle sizing. In variousembodiments, the compositions include, but are not limited to,cross-linked, fatty acid-derived biomaterials (e.g., cross-linked fishoils), and/or combinations of therapeutic agents and cross-linked, fattyacid-derived biomaterials. The compositions can be associated with acryogenic liquid by being directly or indirectly suspended, submerged,surrounded or cooled by a cryogenic liquid.

Suitable fragmentation methods include, but are not limited to,grinding, shearing, shocking, shattering, granulating, pulverizing,shredding, crushing, homogenizing, and/or milling. Suitable means forfragmenting the starting materials into solid particles include, but arenot limited to, mills, e.g., screening mills and impact mills such ashammer mills, and homogenizers, e.g., rotor-stator homogenizers. Thecompositions can be fragmented for a number of cycles, i.e., fragmentingthe compositions for a specified period of time, followed by a specifiedperiod of time in which the compositions are allowed to rest. Forexample the compositions can be fragmented for 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 cycles. In various embodiments, the compositions are fragmentedfrom 4 to 8 cycles.

Therapeutic Agents

In various aspects, a therapeutic agent is loaded into the cross-linked,fatty acid-derived biomaterial before particle formation. In anotheraspect, the cross-linked, fatty acid-derived biomaterial is formed intoa film, fragmented (with or without a cryogenic liquid), the resultingparticles are combined with a therapeutic agent, and theparticle/therapeutic agent composition is further fragmented.Accordingly, the therapeutic agent can be loaded at any time, includingbefore particle formation, during particle formation, or after particleformation. To form the particles, the material, with or without atherapeutic agent, is suspended in a cryogenic liquid, e.g., liquidnitrogen, and fragmented. In various embodiments, the composition isfragmented using a mortar and pestle. If the original startingcomposition did not have a therapeutic agent, therapeutic agent can thenbe added to the resulting particles. To further adjust the particle sizeof the therapeutic agent-containing particles, or to create an emulsionor suspension with the particles, a secondary method, such as acryogrinder can be used.

In various aspects, a therapeutic agent is combined with a fatty acidcompound prior to formation of the film. The resultant film has thetherapeutic agent interspersed throughout the film. The film can then befragmented into particles by any of the methods previously mentioned.

In various aspects, a therapeutic agent is applied in the form of acoating on a stand-alone film. A therapeutic agent can be dissolved inan appropriate solvent (e.g., methanol or ethanol). Thetherapeutic/solvent mixture is applied to the stand-alone film (dipped,sprayed, or brushed onto the film), and the solvent is evaporatedleaving the therapeutic agent loaded into the film. The film is thenfragmented into particles by any of the methods previously mentioned.

In various aspects, a therapeutic agent can be loaded into thefragmented particles during the fragmenting process. The therapeutic canbe added to the cross-linked, fatty acid-derived biomaterial in powderform, with or without a solubilizer, or a non-solvent and fragmentedusing any of the methods previously mentioned. If a solvent ornon-solvent was used, it can be removed, e.g., dried off, or evaporated.The resulting cross-linked, fatty acid-derived biomaterial particles areloaded with the therapeutic agent.

In various aspects, a therapeutic agent is loaded into the cross-linked,fatty acid-derived biomaterial after the material has been fragmented.For example, a cross-linked, fatty acid-derived biomaterial isfragmented, e.g., suspended in liquid nitrogen and ground with a mortarand pestle. The resulting particles of the cross-linked, fattyacid-derived biomaterial are then soaked in a mixture of a therapeuticagent and a solvent. The solvent is then removed, e.g., dried off orevaporated, yielding cross-linked, fatty acid-derived biomaterialparticles loaded with a therapeutic agent. If desired, an additionalmortar and pestle or cryogrind cycle may be used to break up anyclumping.

As utilized herein, the phrase “therapeutic agent(s)” refers to a numberof different drugs or agents available, as well as future agents thatcan be useful. The therapeutic agent component can take a number ofdifferent forms including anti-oxidants, anti-inflammatory agents,anti-coagulant agents, drugs to alter lipid metabolism,anti-proliferatives, anti-neoplastics, tissue growth stimulants,functional protein/factor delivery agents, anti-infective agents,anti-imaging agents, anesthetic agents, therapeutic agents, tissueabsorption enhancers, anti-adhesion agents, germicides, anti-septics,analgesics, prodrugs, and analogs or derivatives thereof and anyadditional desired therapeutic agents such as those listed in Table 1below.

TABLE 1 CLASS EXAMPLES Antioxidants Alpha-tocopherol, lazaroid,probucol, phenolic antioxidant, resveretrol, AGI-1067, vitamin EAntihypertensive Agents Diltiazem, nifedipine, verapamilAntiinflammatory Agents Glucocorticoids (e.g. dexamethazone,methylprednisolone), leflunomide, NSAIDS, ibuprofen, acetaminophen,hydrocortizone acetate, hydrocortizone sodium phosphate,macrophage-targeted bisphosphonates Growth Factor Angiopeptin, trapidil,suramin Antagonists Antiplatelet Agents Aspirin, dipyridamole,ticlopidine, clopidogrel, GP IIb/IIIa inhibitors, abcximab AnticoagulantAgents Bivalirudin, heparin (low molecular weight and unfractionated),wafarin, hirudin, enoxaparin, citrate Thrombolytic Agents Alteplase,reteplase, streptase, urokinase, TPA, citrate Drugs to Alter LipidFluvastatin, colestipol, lovastatin, Metabolism (e.g. statins)atorvastatin, amlopidine ACE Inhibitors Elanapril, fosinopril,cilazapril Antihypertensive Agents Prazosin, doxazosinAntiproliferatives and Cyclosporine, cochicine, mitomycin C,Antineoplastics sirolimus micophenonolic acid, rapamycin, everolimus,tacrolimus, paclitaxel, QP-2, actinomycin, estradiols, dexamethasone,methatrexate, cilostazol, prednisone, cyclosporine, doxorubicin,ranpirnas, troglitzon, valsarten, pemirolast, C-MYC antisense,angiopeptin, vincristine, PCNA ribozyme, 2-chloro-deoxyadenosine Tissuegrowth stimulants Bone morphogeneic protein, fibroblast growth factorPromotion of hollow Alcohol, surgical sealant polymers, polyvinyl organocclusion or particles, 2-octyl cyanoacrylate, hydrogels, thrombosiscollagen, liposomes Functional Protein/Factor Insulin, human growthhormone, estradiols, delivery nitric oxide, endothelial progenitor cellantibodies Second messenger Protein kinase inhibitors targetingAngiogenic Angiopoetin, VEGF Anti-Angiogenic Endostatin Inhibition ofProtein Halofuginone, prolyl hydroxylase inhibitors, Synthesis/ECMformation C-proteinase inhibitors Antiinfective Agents Penicillin,gentamycin, adriamycin, cefazolin, amikacin, ceftazidime, tobramycin,levofloxacin, silver, copper, hydroxyapatite, vancomycin, ciprofloxacin,rifampin, mupirocin, RIP, kanamycin, brominated furonone, algaebyproducts, bacitracin, oxacillin, nafcillin, floxacillin, clindamycin,cephradin, neomycin, methicillin, oxytetracycline hydrochloride,Selenium. Gene Delivery Genes for nitric oxide synthase, human growthhormone, antisense oligonucleotides Local Tissue perfusion Alcohol, H2O,saline, fish oils, vegetable oils, liposomes Nitric oxide Donor NCX4016 - nitric oxide donor derivative of Derivatives aspirin, SNAP GasesNitric oxide, compound solutions Imaging Agents Halogenated xanthenes,diatrizoate meglumine, diatrizoate sodium Anesthetic Agents Lidocaine,benzocaine Descaling Agents Nitric acid, acetic acid, hypochloriteAnti-Fibrotic Agents Interferon gamma -1b, Interluekin - 10Immunosuppressive/ Cyclosporine, rapamycin, mycophenolateImmunomodulatory Agents motefil, leflunomide, tacrolimus, tranilast,interferon gamma-1b, mizoribine Chemotherapeutic Agents Doxorubicin,paclitaxel, tacrolimus, sirolimus, fludarabine, ranpirnase TissueAbsorption Fish oil, squid oil, omega 3 fatty acids, Enhancers vegetableoils, lipophilic and hydrophilic solutions suitable for enhancingmedication tissue absorption, distribution and permeation Anti-AdhesionAgents Hyaluronic acid, human plasma derived surgical sealants, andagents comprised of hyaluronate and carboxymethylcellulose that arecombined with dimethylaminopropyl, ethylcarbodimide, hydrochloride, PLA,PLGA Ribonucleases Ranpirnase Germicides Betadine, iodine, slivernitrate, furan derivatives, nitrofurazone, benzalkonium chloride,benzoic acid, salicylic acid, hypochlorites, peroxides, thiosulfates,salicylanilide Antiseptics Selenium Analgesics Bupivicaine, naproxen,ibuprofen, acetylsalicylic acid

The therapeutic agent can be an active agent as contained in thecross-linked, fatty acid-derived biomaterial. Pharmaceuticallyacceptable salts, esters, or prodrugs of the therapeutic agent are alsosuitable for use. In various embodiments, the cross-linked, fattyacid-derived biomaterial itself comprises the therapeutic agent.

In various embodiments, the therapeutic agent comprises an mTORtargeting compound. The term “mTOR targeting compound” refers to anycompound which modulates mTOR directly or indirectly. An example of an“mTOR targeting compound” is a compound that binds to FKBP 12 to form,e.g., a complex, which in turn inhibits phosphoinositide (PI)-3 kinase,that is, mTOR. In various embodiments, mTOR targeting compounds inhibitmTOR. Suitable mTOR targeting compounds include, for example, rapamycinand its derivatives, analogs, prodrugs, esters and pharmaceuticallyacceptable salts thereof.

Calcineurin is a serine/threonine phospho-protein phosphatase and iscomposed of a catalytic (calcineurin A) subunit and a regulatory(calcineurin B) subunit (about 60 and about 18 kDa, respectively). Inmammals, three distinct genes (A-alpha, A-beta, A-gamma) for thecatalytic subunit have been characterized, each of which can undergoalternative splicing to yield additional variants. Although mRNA for allthree genes appears to be expressed in most tissues, two isoforms(A-alpha and A-beta) are most predominant in brain.

The calcineurin signaling pathway is involved in immune response as wellas apoptosis induction by glutamate excitotoxicity in neuronal cells.Low enzymatic levels of calcineurin have been associated withAlzheimer's disease. In the heart or in the brain calcineurin also playsa key role in the stress response after hypoxia or ischemia.

Substances which are able to block the calcineurin signal pathway can besuitable therapeutic agents. Examples of such therapeutic agentsinclude, but are not limited to, FK506, tacrolimus, cyclosporin andinclude derivatives, analogs, esters, prodrugs, pharmaceuticallyacceptably salts thereof, and conjugates thereof which have or whosemetabolic products have the same mechanism of action. Further examplesof cyclosporin include, but are not limited to, naturally occurring andnon-natural cyclosporins prepared by total- or semi-synthetic means orby the application of modified culture techniques. The class comprisingcyclosporins includes, for example, the naturally occurring CyclosporinsA through Z, as well as various non-natural cyclosporin derivatives,artificial or synthetic cyclosporin derivatives. Artificial or syntheticcyclosporins can include dihydrocyclosporins, derivatized cyclosporins,and cyclosporins in which variant amino acids are incorporated atspecific positions within the peptide sequence, for example,dihydro-cyclosporin D.

In various embodiments, the therapeutic agent comprises one or more of amTOR targeting compound and a calcineurin inhibitor. In variousembodiments, the mTOR targeting compound is a rapamycin or a derivative,analog, ester, prodrug, pharmaceutically acceptably salts thereof, orconjugate thereof which has or whose metabolic products have the samemechanism of action. In various embodiments, the calcineurin inhibitoris a compound of Tacrolimus, or a derivative, analog, ester, prodrug,pharmaceutically acceptably salts thereof, or conjugate thereof whichhas or whose metabolic products have the same mechanism of action or acompound of Cyclosporin or a derivative, analog, ester, prodrug,pharmaceutically acceptably salts thereof, or conjugate thereof whichhas or whose metabolic products have the same mechanism of action.

In various embodiments, the therapeutic agent comprises an anti-adhesiveagent. As used herein, the term “anti-adhesion agent” refers to anycompound that prevents adhesions or accretions of body tissues formed inresponse to injury of various kinds, e.g., surgery, infection,chemotherapy, radiation. Anti-adhesion agents include, but are notlimited to, hyaluronic acid, human plasma derived surgical sealants, andagents comprised of hyaluronate and carboxymethylcellulose that arecombined with dimethylaminopropyl, ethylcarbodimide, hydrochloride, PLA,and/or PLGA.

In various embodiments, the therapeutic agent comprises anantiproliferative and/or an antineoplastic agent. The term“antiproliferative/antineoplastic agent” as used herein refers to anycompound which inhibits or prevents the growth or development of cells,e.g., smooth muscle cells, or malignant cells. Suitableantiproliferative and antineoplastic agents include, but are not limitedto, paclitaxel or its derivatives, analogs, esters, prodrugs, andpharmaceutically acceptable salts thereof.

A therapeutically effective amount refers to that amount of a compoundsufficient to result in amelioration of symptoms, e.g., treatment,healing, prevention or amelioration of the relevant medical condition,or an increase in rate of treatment, healing, prevention or ameliorationof such conditions. When applied to an individual active ingredient,administered alone, a therapeutically effective amount refers to thatingredient alone. When applied to a combination, a therapeuticallyeffective amount can refer to combined amounts of the active ingredientsthat result in the therapeutic effect, whether administered incombination, serially or simultaneously. In various embodiments, whereformulations comprise two or more therapeutic agents, such formulationscan be described as a therapeutically effective amount of compound A forindication A and a therapeutically effective amount of compound B forindication B, such descriptions refer to amounts of A that have atherapeutic effect for indication A, but not necessarily indication B,and amounts of B that have a therapeutic effect for indication B, butnot necessarily indication A.

Actual dosage levels of the active ingredients in the compositionsprovided herein can be varied so as to obtain an amount of the activeingredients which is effective to achieve the desired therapeuticresponse without being unacceptably toxic. The selected dosage levelwill depend upon a variety of pharmacokinetic factors including theactivity of the particular therapeutic agent (drug) employed, or theester, salt or amide thereof, the mechanism of drug action, the time ofadministration, the drug release profile of the coating, the rate ofexcretion of the particular compounds being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compounds employed, and like factorsknown in the medical arts.

As used herein, the term “solid therapeutic agent” refers to therapeuticagents in solid form, i.e., not liquids or gases.

In one embodiment, the therapeutic agent is stabilized. The biologicallyactive agent can be stabilized against degradation, loss of potencyand/or loss of biological activity, all of which can occur duringformation of the particles having the biologically active agentdispersed therein, and/or prior to and during in vivo release of thebiologically active agent from the particles. In one embodiment,stabilization can result in a decrease in the solubility of thebiologically active agent, the consequence of which is a reduction inthe initial release of the biologically active agent, in particular,when release is from particles for sustained release of the biologicallyactive agent. In addition, the period of release of the biologicallyactive agent from the particles can be prolonged.

Stabilization of the biologically active agent can be accomplished, forexample, by the use of a stabilizing agent or a specific combination ofstabilizing agents. “Stabilizing agent,” as that term is used herein, isany agent which binds or interacts in a covalent or non-covalent manneror is included with the biologically active agent.

In another embodiment, the stabilizing agent can be vitamin E. It shouldbe noted that as utilized herein to describe the present invention, theterm “vitamin E” and the term “alpha-tocopherol,” are intended to referto the same or substantially similar substance, such that they areinterchangeable and the use of one includes an implicit reference toboth. Further included in association with the term vitamin E are suchvariations including but not limited to one or more of alpha-tocopherol,beta-tocopherol, delta-tocopherol, gamma-tocopherol, alpha-tocotrienol,beta-tocotrienol, delta-tocotrienol, gamma-tocotrienol, alpha-tocopherolacetate, beta-tocopherol acetate, gamma-tocopherol acetate,delta-tocopherol acetate, alpha-tocotrienol acetate, beta-tocotrienolacetate, delta-tocotrienol acetate, gamma-tocotrienol acetate,alpha-tocopherol succinate, beta-tocopherol succinate, gamma-tocopherolsuccinate, delta-tocopherol succinate, alpha-tocotrienol succinate,beta-tocotrienol succinate, delta-tocotrienol succinate,gamma-tocotrienol succinate, mixed tocopherols, vitamin E TPGS,derivatives, analogs and pharmaceutically acceptable salts thereof.

The therapeutic agents that can be used with the fatty acid-derivedparticles of the invention can also include antimicrobial agents,including, antivirals, antibiotics, antifungals and antiparasitics.Specific antimicrobial agents that can be used with the the fattyacid-derived particles of the invention of the invention includeGentamicin Sulfate, Penicillin G, ephalothin, Ampicillin, Amoxicillin,Augmentin, Aztreonam, Imipenem, Streptomycin, Gentamicin, Vancomycin,Clindamycin, Erythromycin, Azithromycin, Polymyxin, Bacitracin,Amphotericin, Nystatin, Rifampicin, Tetracycline, Doxycycline,Chloramphenicol, Nalidixic acid, Ciprofloxacin, Sulfanilamide,Gantrisin, Trimethoprim, Isoniazid (INH), para-aminosalicylic acid(PAS), Minocycline, and silver compounds, such as silver nitrate, silverbenzoate, and nano-silver.

“Sustained release,” as that term is used herein, is a release of thebiologically active agent from the fatty acid-based particles thatoccurs over a period which is longer than the period during which abiologically significant amount of the active agent would be availablefollowing direct administration of the active agent, e.g., a solution orsuspension of the active agent. In various embodiments, a sustainedrelease is a release of the biologically active agent which occurs overa period of at least about one day such as, for example, at least about2, 4, 6, 8, 10, 15, 20, 30, 60, or at least about 90 days. A sustainedrelease of the active agent can be a continuous or a discontinuousrelease, with relatively constant or varying rates of release. Thecontinuity of release and level of release can be affected by thebiologically active agent loading, and/or selection of excipients toproduce the desired effect.

“Sustained release,” as used herein, also encompasses “sustained action”or “sustained effect.” “Sustained action” and “sustained effect,” asthose terms are used herein, refer to an increase in the time periodover which the biologically active agent performs its therapeutic,prophylactic and/or diagnostic activity as compared to an appropriatecontrol. “Sustained action” is also known to those experienced in theart as “prolonged action” or “extended action.”

Uses of Particles

The fatty acid-based particles, either alone or in combination with anadditional therapeutic agent, can be used in vivo in a variety of ways.For example, the particles can be used as a dry powder in inhalertreatments, as an aqueous dispersion for intravenous use, or in acapsule for oral delivery. The particles can also be applied topically.In various embodiments, the particles can be sprinkled over a desiredlocation in vivo. The particles can be used with another medical device,e.g., a graft or mesh. The particles can also be used without anothermedical device, e.g., as anti-adhesion particles.

The fatty acid particles of the invention can be used for the preventionof surgical adhesion, as they can minimize surgical adhesions withoutcausing inflammation by being placed in a desired location. Thus, forexample, the particles can be sprinkled over a desired location in vivo,e.g., an area of a surgical procedure, for purposes of preventingsurgical adhesions.

Also provided herein is a fatty acid-based material (in particle form orparticles pressed into a film) suitable for treating or preventingdisorders related to vascular injury and/or vascular inflammation. Thefatty acid-based material (in particle form or particles pressed into afilm) can also be used to treat or prevent injury to tissue, e.g., softtissue. In another embodiment, the source of the fatty acid for thematerial is an oil, such as fish oil.

In general, four types of soft tissue are present in humans: epithelialtissue, e.g., the skin and the lining of the vessels and many organs;connective tissue, e.g., tendons, ligaments, cartilage, fat, bloodvessels, and bone; muscle, e.g., skeletal (striated), cardiac, orsmooth; and nervous tissue, e.g., brain, spinal chord and nerves. Thefatty acid-based material of the invention (in particle form orparticles pressed into a film) can be used to treat injury to these softtissue areas. Thus, in one embodiment, the fatty acid-based materials ofthe invention in particle form or particles pressed into a film) can beused for promotion of proliferation of soft tissue for wound healing.Furthermore, following acute trauma, soft tissue can undergo changes andadaptations as a result of healing and the rehabilitative process. Suchchanges include, by are not limited to, metaplasia, which is conversionof one kind of tissue into a form that is not normal for that tissue;dysplasia, with is the abnormal development of tissue; hyperplasia,which is excessive proliferation of normal cells in the normal tissuearrangement; and atrophy, which is a decrease in the size of tissue dueto cell death and resorption or decreased cell proliferation.Accordingly, the fatty acid-based material of the invention (in particleform or particles pressed into a film) can be used for the diminishmentor alleviation of at least one symptom associated with or caused byacute trauma in soft tissue.

In one embodiment, as described below, the fatty acid-based material (inparticle form or particles pressed into a film) can be used, forexample, to prevent tissue adhesion. The tissue adhesion can be a resultof blunt dissection. Blunt dissection can be generally described asdissection accomplished by separating tissues along natural cleavagelines without cutting. Blunt dissection is executed using a number ofdifferent blunt surgical tools, as is understood by those of ordinaryskill in the art. Blunt dissection is often performed in cardiovascular,colo-rectal, urology, gynecology, upper GI, and plastic surgeryapplications, among others.

After the blunt dissection separates the desired tissues into separateareas, there is often a need to maintain the separation of thosetissues. In fact, post surgical adhesions can occur following almost anytype of surgery, resulting in serious postoperative complications. Theformation of surgical adhesions is a complex inflammatory process inwhich tissues that normally remain separated in the body come intophysical contact with one another and attach to each other as a resultof surgical trauma.

It is believed that abdominal adhesions are formed when bleeding andleakage of plasma proteins from damaged tissue deposit in the abdominalcavity and form what is called a fibrinous exudate. Fibrin, whichrestores injured tissues, is sticky, so the fibrinous exudate may attachto adjacent anatomical structures in the abdomen. Post-traumatic orcontinuous inflammation exaggerates this process, as fibrin depositionis a uniform host response to local inflammation. This attachment seemsto be reversible during the first few days after injury because thefibrinous exudates go through enzymatic degradation caused by therelease of fibrinolytic factors, most notably tissue-type plasminogenactivator (t-PA). There is constant play between t-PA andplasminogen-activator inhibitors. Surgical trauma usually decreases t-PAactivity and increases plasminogen-activator inhibitors. When thishappens, the fibrin in the fibrinous exudate is replaced by collagen.Blood vessels begin to form, which leads to the development of anadhesion. Once this has occurred, the adhesion is believed to beirreversible. Therefore, the balance between fibrin deposition anddegradation during the first few days post-trauma is critical to thedevelopment of adhesions (Holmdahl L. Lancet 1999; 353: 1456-57). Ifnormal fibrinolytic activity can be maintained or quickly restored,fibrous deposits are lysed and permanent adhesions can be avoided.Adhesions can appear as thin sheets of tissue or as thick fibrous bands.

Often, the inflammatory response is also triggered by a foreignsubstance in vivo, such as an implanted medical device. The body seesthis implant as a foreign substance, and the inflammatory response is acellular reaction to wall off the foreign material. This inflammationcan lead to adhesion formation to the implanted device; therefore amaterial that causes little to no inflammatory response is desired.

Thus, the fatty acid-based material (in particle form or particlespressed into a film) can be used as a barrier to keep tissues separatedto avoid the formation of adhesions, e.g., surgical adhesions.Application examples for adhesion prevention include abdominalsurgeries, spinal repair, orthopedic surgeries, tendon and ligamentrepairs, gynecological and pelvic surgeries, and nerve repairapplications. The fatty acid-based material may be applied over thetrauma site (e.g., in the case of particles, in may be sprinkled) or(e.g., in the case of the pressed film) wrapped around the tissue ororgan to limit adhesion formation. The addition of therapeutic agents tothe fatty acid-based material (in particle form or particles pressedinto a film) used in these adhesion prevention applications can beutilized for additional beneficial effects, such as pain relief orinfection minimization. Other surgical applications of the fattyacid-based pressed film may include using the film as a dura patch,buttressing material, internal wound care (such as a graft anastomoticsite), and internal drug delivery system. The fatty acid-based material(in particle form or particles pressed into a film) may also be used inapplications in transdermal, wound healing, and non-surgical fields. Thefatty acid-based material (in particle form or particles pressed into afilm) may be used in external wound care, such as a treatment for burnsor skin ulcers. The fatty acid-based material (in particle form orparticles pressed into a film) may be used without any therapeutic agentas a clean, non-permeable, non-adhesive, non-inflammatory,anti-inflammatory dressing, or the fatty acid-based material (inparticle form or particles pressed into a film) may be used with one ormore therapeutic agents for additional beneficial effects. When used inany of the aforementioned methods, the fatty acid-based material (inparticle form or particles pressed into a film) may or may not beassociated with a therapeutic agent.

The process of wound healing involves tissue repair in response toinjury and it encompasses many different biologic processes, includingepithelial growth and differentiation, fibrous tissue production andfunction, angiogenesis, and inflammation. Accordingly, the fattyacid-based material (in particle form or particles pressed into a film)provides an excellent material suitable for wound healing applications.

Also, the administration of a variety of therapeutic agents using theparticles of the invention is advantageous, as the particles have anincreased surface area and increased bioavailability over other carrieragents. Furthermore, the particles of the invention will not induce aninflammatory response in vivo.

In various embodiments, the particles formed by the methods providedherein can be mixed with an oil to create an emulsion, or insolubleformulation. The oil can be cured or uncured. Such a formulation can beused as an extended release coating on a medical device, e.g., a graftor a mesh. The formulation can also be spread onto a substrate surfaceand allowed to cure for a period of time to cure the layer of theformulation, without degrading the drug.

The particles alone or with an additional therapeutic agent can also besprinkled onto a stand alone film comprised of a cross-linked, fattyacid-derived biomaterial prior to the film being cured, e.g., placed inan oven. The resulting textured film can then be used in vivo.

The fatty acid particles can be cryoground with a fatty acid compound,e.g., a fish oil, which will substantially thicken the fatty acidcompound to a gel-like substance. The viscosity is directly related tothe ratio of particles to the fatty acid compound, as well as the lengthof cure of the fatty acid compound mixture.

Modulated Healing

Also provided herein are fatty acid based-particles suitable forachieving modulated healing in a tissue region in need thereof, whereinthe composition is administered in an amount sufficient to achieve saidmodulated healing. In one embodiment, the source of the fatty acid forthe particles is an oil, such as fish oil.

Modulated healing can be described as the in-vivo effect observedpost-implant (e.g., from sprinkling) in which the biological response isaltered resulting in a significant reduction in foreign body response.As utilized herein, the phrase “modulated healing” and variants of thislanguage generally refers to the modulation (e.g., alteration, delay,retardation, reduction, detaining) of a process involving differentcascades or sequences of naturally occurring tissue repair in responseto localized tissue injury, substantially reducing their inflammatoryeffect. Modulated healing encompasses many different biologic processes,including epithelial growth, fibrin deposition, platelet activation andattachment, inhibition, proliferation and/or differentiation, connectivefibrous tissue production and function, angiogenesis, and several stagesof acute and/or chronic inflammation, and their interplay with eachother. For example, the fatty acids described herein can alter, delay,retard, reduce, and/or detain one or more of the phases associated withhealing of vascular injury caused by medical procedures, including, butnot limited to, the inflammatory phase (e.g., platelet or fibrindeposition), and the proliferative phase. In one embodiment, “modulatedhealing” refers to the ability of a fatty acid-based particle to alter asubstantial inflammatory phase (e.g., platelet or fibrin deposition) atthe beginning of the tissue healing process. As used herein, the phrase“alter a substantial inflammatory phase” refers to the ability of theparticle to substantially reduce the inflammatory response at an injurysite. In such an instance, a minor amount of inflammation may ensue inresponse to tissue injury, but this level of inflammation response,e.g., platelet and/or fibrin deposition, is substantially reduced whencompared to inflammation that takes place in the absence of the fattyacid-based particle.

In another non-binding example, the modulated healing effect can beattributed to the modulation (e.g., alteration, delay, retardation,reduction, detaining) of signaling between the cells and proteins thatcompose the vessel wall and various components of the bloodstream thatwould otherwise initiate the vascular healing process. Stateddifferently, at the site of vascular injury, the particles can modulatethe interaction of cells of the vessel wall, such as endothelial cellsand/or smooth muscle cells, with other cells and/or proteins of theblood that would otherwise interact with the damaged cells to initiatethe healing process. Additionally, at the site of vascular injury, theparticles can modulate the interaction of proteins of the vessel wallwith other cells and/or proteins of the blood, thereby modulating thehealing process. When used in any of the aforementioned methods, thefatty acid-based material (in particle form or particles pressed into afilm) may or may not be associated with a therapeutic agent.

The fatty acid derived material (in particle form or particles pressedinto a film) can be designed to maintain its integrity for a desiredperiod of time, and then begin to hydrolyze and be absorbed into thetissue that it is surrounded by. Alternatively, the fatty acid derivedmaterial (in particle form or particles pressed into a film) can bedesigned such that, to some degree, it is absorbed into surroundingtissue immediately after the fatty acid derived material (in particleform or particles pressed into a film) is inserted in the subject.Depending on the formulation of the particles, it can be completelyabsorbed into surrounding tissue within a time period of 1 day to 24months, e.g., 1 week to 12 months, e.g., 1 month to 10 months, e.g., 3months to 6 months.

Medical Devices

FIGS. 13A-13E illustrate some forms of medical devices mentioned abovethat can be combined with the particles. In one embodiment, theparticles can be used to coat the medical devices. FIG. 13A shows agraft 50 with the particles 10 coupled or adhered thereto. FIG. 13Bshows a catheter balloon 52 with the particles 10 coupled or adheredthereto. FIG. 13C shows a stent 54 with the particles 10 coupled oradhered thereto. FIG. 13D illustrates a stent 10 in accordance with oneembodiment of the present invention. The stent 10 is representative of amedical device that is suitable for having particles applied thereon toeffect a therapeutic result. The stent 10 is formed of a series ofinterconnected struts 12 having gaps 14 formed therebetween. The stent10 is generally cylindrically shaped. Accordingly, the stent 10maintains an interior surface 16 and an exterior surface 18. FIG. 13Eillustrates a coated surgical mesh, represented as a biocompatible meshstructure 10, in accordance with one embodiment of the presentinvention. The biocompatible mesh structure 10 is flexible, to theextent that it can be placed in a flat, curved, or rolled configurationwithin a patient. The biocompatible mesh structure 10 is implantable,for both short term and long term applications. Depending on theparticular formulation of the biocompatible mesh structure 10, thebiocompatible mesh structure 10 will be present after implantation for aperiod of hours to days, or possibly months, or permanently.

Each of the medical devices illustrated, in addition to others notspecifically illustrated or discussed, can be combined with theparticles of the invention using the methods described herein, orvariations thereof. Accordingly, the present invention is not limited tothe example embodiments illustrated. Rather the embodiments illustratedare merely example implementations of the present invention.

EXAMPLES

Various aspects and embodiments of the present invention are furtherdescribed by way of the following Examples. The Examples are offered byway of illustration and not by way of limitation.

1. Fatty Acid-Based Particles Soaked in Rapamycin/Solvent Combination.

A 0.005 inch thin film was made according to the methods describedherein. Briefly, fish oil was thickened using heat and oxygen until thedesired viscosity was reached. This was accomplished by heating thenative fish oil to 93° C. and bubbling oxygen into the heated oil withconstant stirring at a rate of 5 standard cubic feet per minute until aviscosity of 20,000 cP @22° C. was reached (a total of 15 hours). Thethickened oil was cast onto PTFE coated plates using an adjustablecasting knife set at 0.01″. The cast oil was UV treated for 15 minutesand then placed into a 200° F. oven for 24 hours to achieve across-linked, fatty acid-derived biomaterial film.

The film was then converted into particles either by placing the filminto a mortar, covering it with liquid nitrogen and using the pestle togrind it into particle form or by cryogrinding it into particle form.200 mg each of mortar and pestle film particles and cryoground filmparticles were soaked in 1 ml of 25 mg/ml rapamycin in ethanol for about5 minutes. An aluminum weigh pan was lined with 0.45 um filter paper.The particles were poured into the weigh pan and allowed to air dry forabout 10 minutes and then placed under vacuum to remove any residualsolvent.

40 mg of particles from each grinding method were placed into 3 vialsfor HPLC analysis: one for dissolution, and two for extraction.Dissolution was conducted in 0.01M PBS. Samples were taken daily.Extraction showed 600 ug RAP per 40 mg of particles using both themortar and pestle and the cryoground method. The elution profiles areshown in FIG. 2

2. A Thin Film Loaded by Rapamycin/Solvent Spray Prior to ParticleFormation

A 0.005″ thick non-polymeric cross-linked film was made according to themethods described herein. The film was then cut into two 3×5″ filmsections. The film sections were sprayed with 25 mg/mlrapamycin/methanol, 4 layers on each side. The films were then groundusing a mortar and pestle with liquid nitrogen. 40 mg of particles werethen placed into 2 eppendorf tubes for HPLC analysis, one fordissolution and one for extraction. The remaining ground particles werecryoground for eight 2 minute cycles at 30 impacts per second. 40 mg ofthe cryoground particles were placed into two eppendorf tubes for HPLCanalysis, one for dissolution and one for extraction. Extraction showed:242 ug RAP/40 mg mortar and pestle particles; 229 ug RAP/40 mgcryoground particles.

Dissolution was conducted in 0.01M PBS. Samples were taken daily. FIG. 3shows the elution profiles of each group of particles. Both the mortarand pestle particles and the cryoground particles elute similar amountsof RAP by the end of the dissolution, but at different rates.

3. Film Particles Soaked in Cyclosporine A/Solvent

Two grams of particles derived from cryoground non-polymericcross-linked film were soaked in 5 ml of 25 mg/ml Cyclosporin A inmethanol for approximately 15 minutes. An aluminum weigh pan was linedwith 0.45 um filter paper. The particles were poured into the weigh panand allowed to air dry in the back of the fume hood for 3 hours atambient conditions. The films were then ground using a mortar and pestlewith liquid nitrogen. 40 mg of particles were placed into 4 eppendorftubes for HPLC analysis, two for dissolution and two for extraction. Theparticles were extracted using methanol. 40 mg of particles had anaverage loading of 170 ug of Cyclosporin A. Dissolution was conducted in0.01M PBS. FIG. 4 shows the elution profile of the particles, the finaldata point is the final extraction value of CSA from the particles.

4. A Thin Film Loaded by Marcaine/Solvent Spray Prior to being Groundinto Particles

A thin film comprised of a cross-linked, fatty acid-derived biomaterial,formed from fish oil, approximately 8¼ by 4¾″, was sprayed, 3 layers oneach side, with 25 mg/ml Marcaine/methanol. The film was then groundusing a mortar and pestle with liquid nitrogen. 50 mg of particles wereplaced into 4 eppendorf tubes for HPLC analysis, two for dissolution andtwo for extraction. The particles were extracted using acetonitrile. 50mg of particles had an average loading of 180 ug Marcaine. Dissolutionwas conducted in 0.01M PBS and samples were taken daily. FIG. 5 showsthe elution profile for these particles.

5. Use of Particles Comprising a Cross-Linked, Fatty Acid-DerivedBiomaterial for Drug Delivery in a Multi-Lumen Vascular Graft

A multi-lumen vascular graft was manufactured using the methodsdescribed in U.S. Pat. No. 5,411,550. The multi-lumen graft comprises aprimary lumen and at least one secondary lumen separated from theprimary lumen by a wall sufficiently permeable to permit a bioactivematerial disposed into the secondary lumen to diffuse through the wallinto the main lumen or through the wall to the advential surface.

A multi-lumen graft with a primary lumen of 6 mm inside diameter and 0.6mm secondary lumen is selected. Particles of a cross-linked, fattyacid-derived biomaterial, formed from fish oil, were mixed with asolvent, e.g., acetone, to swell the particles. The swelled particlesare then loaded into a syringe with a 22 gauge blunt tip needleattached. The needle is placed into one of the secondary lumens, andpressure is then applied to the plunger on the syringe assembly to forcethe swelled particles through the needle into the secondary lumen untilit is substantially completely filled. This process is repeated forthree remaining secondary lumens. Once the lumens were filled, the graftis placed into a 200° F. oven for 30 minutes to evaporate the solvent.

6. Film Particles Cryoground with Drug Powder: Directly, with MeOH andwith Hexane

Fatty acid particles were prepared by grinding a thin film of fish oil(prepared as described in Example 1) using a mortar and pestle withliquid nitrogen. Two grams of the mortar and pestled particles werecryoground in the Spex Certiprep Cryomill (Model 6750) 3 different ways:

1. with 227 mg RAP and 4 ml Hexane (RAP non-solubilizing solvent);

2. with 224 mg RAP and 4 ml MeOH (RAP solubilizing solvent); and

3. with 223 mg RAP (dry).

All formulations were cryoground for eight 2 minute cycles with 2 minuterest between cycles at an impactor speed of 15. Samples containingsolvent were dried down using a vacuum chamber over the weekend (forconvenience rather than necessity). The samples that were originallyground with solvent were re-cryoground to remove clumping. Approximately50 mg of sample was each added to 4 eppendorf tubes (total of 12 tubes)for dissolution and extraction. Extraction was performed using 1 ml ofan acetonitrile, acetic acid combination. All three methods had andaverage loading of 4 mg RAP per 50 mg of particles. Dissolution wasconducted in 0.01 M PBS and samples were taken daily. FIG. 6 shows theelution profile of the particles (the final data point is the finalextraction values of RAP from the particles).

7. Fatty Acid-Based Particles: Cyclosporine Added to the Particle PhaseVs. the Oil Phase

Fatty acid particles are prepared by grinding a thin film of fish oil(prepared as described herein) using a mortar and pestle with liquidnitrogen, followed by cryogrinding (8 cycles, 2 minute cool, 2 minutecryogrind, 30 impacts/second). 1.2 g cryoparticles are combined with 2.8g fish oil in a cryovial. The components are then cryoground (8 cycles,2 minute cool, 2 minute cryogrind, 30 impacts/second). The result is agel substance.

Optionally, therapeutic agents can be added to the gel either in theparticle phase, the oil phase, or both. This example compares thetherapeutic loaded into the particle phase vs. the oil phase.

Particle Phase:

1.5 g mortar and pestle ground particles

27.6 mg Cyclosporine A

Cryogrind these together as described above

Add: 3.5 g native fish oil

Cryogrind these together as described above

Oil Phase:

3.5 g native fish oil

28.5 mg Cyclosporine A

Heat to solubilize drug

Add: 1.5 g cryoparticles

Cryogrind these together as described above

The plot shown in FIG. 7 shows the average dissolution of two 100 mgsamples in 0.1M PBS. The final data point is the final extraction.

8. CSA Added to Fatty Acid Compound to Form a Thin Film which can beGround into Fatty Acid Particles Containing CSA

Cyclosporine A was directly added to the thickened fish oil at 3.6%, andthe resulting composition was solubilized by heating the components at200° F. for 10 minutes. The oil was then cast into a thin film and curedfor 24 hours at 200° F. This film with CSA dispersed within can then beground into particles. The plot shown in FIG. 8 shows the averagedissolution curve of 1×3″ film samples prior to particle formation. Eachsample contained approximately 1300 ug active CSA (determined byextraction). The last data point is the final extraction. With particleformation it is thought to have more complete release during thedissolution phase due to the increase in surface area.

9. A Pressed Film Comprised of Cured Fish Oil Film Particles Cryogroundwith Gentamicin Sulfate

Fatty acid particles were formed by grinding a cured fish oil film usingliquid nitrogen and a mortar and pestle. These particles were thencryoground in the Spex Certiprep Cryomill with 6% Gentamycin Sufate,with a combined particle mass of approximately 3 grams. The formulationwas cryoground for eight 2 minute cycles, with 2 minutes of coolingbetween each cycle, and at a rate of 15 impacts per second. After theparticles came to room temperature, they were again ground using liquidnitrogen and a mortar and pestle to separate particles that had clumpedtogether after cryogrinding. The particles were then formed into a thinfilm using a Carver Press with platen block temperatures of 80° C. and apressure of 1 ton for 4 minutes. The resulting product was a thin,solid, translucent film.

FIG. 9 shows the elution profile of the film in 0.1M Phosphate bufferthat was carried out for 6 days (144 hours) in a 37° C. shaker table.Although the experiment was stopped at day 6, the therapeutic agent wasstill releasing at the 6 day timepoint. The total film mass was 197.8 mgwith a theoretical gravimetric Gentamicin Sulfate loading of 12 mg. Asof 6 days of dissolution, 18.5% of the gravimetric Gentamicin Sulfatehad been released.

10. Zone of Inhibition of a Pressed Film Comprised of Cured Fish OilFilm Particles Cryoground with Gentamicin Sulfate

In addition to dissolution, an in-vitro zone of inhibition assay wasperformed on 0.5 inch diameter disks to show efficacy against platesinoculated with staph aureus. The zone of inhibition is seen as anantibacterial ring surrounding sample disks where the Gentamicin Sulfatehas diffused into the agar. The prepared samples were stored at 37° C.and transferred to a new plate of staph aureus daily. The Total ZoneSize was measured across the entire diameter of the antibacterial ring,which included the diameter of the sample disk. The data displayed onthe bar graph is the difference between Total Zone Size and the SampleSize, resulting in only the size of the actual Zone of Inhibition.

Two sample types were compared with a Zone of Inhibition assay. Thefirst sample type is the pressed particle/Gentamycin Sulfate film thatwas produced in the same manner as mentioned in the dissolution sampledescription above. The average gravimetric Gentamicin Sulfate loading ofn=3 0.5 inch diameter disks was 480 ug/cm². The other sample type wasmade by cryogrinding Gentamicin Sulfate directly with native fish oiland then casting that liquid coating onto bare polypropylene mesh. Thesample is exposed to 200° F. for 40 hours to achieve a solid, curedcoating. The average gravimetric Gentamicin Sulfate loading of n=2 0.5inch diameter disks was 406 ug/cm².

As seen in FIG. 10, the cured mesh samples with Gentamicin Sulfateshowed efficacy for two days against staph aureus. A 120 ug Gentamycinstandard disk was run alongside the cured mesh samples to ensure thatthe assay was not problematic. In general, these standard disks areefficacious for about 3 days, which was the case for this experiment.The standard shows that the reason the cured mesh samples stoppedexhibiting a zone was not a function of the assay, but a function of thesamples themselves. In contrast, the pressed particle film withGentamicin Sulfate has shown activity against staph aureus for 11 daysin agar. The pressed particle samples are still being carried out inzone of inhibition, and the experiment is not complete at the 11 daytime point. Although both sample types were made with comparable amountsof Gentamicin Sulfate, the activity is dependent on how the drug isloaded into the fish oil coating.

11. Film Comprised of Cured Fish Oil Film Particles Cryoground withGentamicin Sulfate Pressed onto Polypropylene Mesh

Fatty acid particles were formed by grinding a cured fish oil film usingliquid nitrogen and a mortar and pestle. These particles were thencryoground with 14% Gentamycin Sufate, with a combined particle mass ofapproximately 3 grams. The formulation was cryoground in the SpexCertiprep Cryomill for eight 2 minute cycles, with 2 minutes of coolingbetween each cycle, and at a rate of 15 impacts per second. After theparticles came to room temperature, they were again ground using liquidnitrogen and a mortar and pestle to separate particles that had clumpedtogether after cryogrinding. The particles were then formed into a thinfilm using a Carver Press with platen block temperatures of 80° C. and apressure of 1 ton for 4 minutes. The resulting product was a thin,solid, translucent film. That film was pressed onto one side of a 1″×1″polypropylene mesh using the Carver Press with platen block temperaturesof 90° C. and a pressure of 400 lbs for 6 minutes.

FIG. 11 shows the elution profile of an average of two mesh-film samplesin 0.1M Phosphate buffer that was carried out for 20 days (480 hours) ina 37° C. shaker table. The samples have an average theoreticalgravimetric Gentamicin Sulfate loading of 5020 ug/cm². As of 20 days ofdissolution, an average of 40.3% of the gravimetric Gentamicin Sulfatehas been released.

12. Prevention of Peridural Fibrosis Using a Fish Oil Particle in theRabbit Spinal Model

The purpose of this study was to test the efficacy of fish oil particlesin the reduction of adhesion formation and biocompatibility at 28 daysin a rabbit model of peridural fibrosis.

The particle gel formulation was made by first creating fatty acidparticles by cryogrinding a standard 24-hour thin film (as explained inExample 1) using the Spex Certiprep Cryomill, eight two-minute cycleswith two-minute cool in between at 30 impacts/second. The particle gelformulation was then prepared by cryogrinding 30% b/w particles innative fish oil using the same cycle parameters.

New Zealand White rabbits were used in the study. A 2-level laminectomywas performed between levels L6 and L4. Using a bur, a 10 mm×5 mm defectwas outlined on the lamina of level L5. Drilling was carried out to thelevel of L4. A similar defect was made on level L6. The dorsal surfaceof the dura was lightly abraded with a “ball” of 4″×4″ sterile gauze(clamped in a pair of hemostats) for a period of 2 minutes to createabrasion trauma on the site of the bone defect.

After conclusion of the surgical procedure the particle gel was placedat the site of the defect to fill the space in the site to receivetherapy (randomized to cephalic or caudal in each rabbit). The controlsite received surgery only.

Twenty eight days post-surgery, the rabbit was euthanized, and thedefect was examined based on the appearance of the surrounding tissues,the amount of blood on the surgical site and the amount of bone thatregenerated on the surgical site. No differences between control andtreated sites were observed for any groups. The vertebra were cut fromeach end of the defect, treated and sent for histological preparation.

Histologic Evaluation:

Prepared slides were evaluated microscopically. The slides were numberedto blind the evaluator. The sections were evaluated for theeffectiveness of the treatment by estimating the amount of epiduralfibrosis in histological slides of the healed defect. This wasdetermined by estimating the amount of fibrosis specifically present inthe epidural space and fibrosis that was attached to the dura. Theamount of fibrosis attached to the dura was determined for each sectionusing the following scoring system:

0 No fibrosis adherent to dura

1% to 30% of dura at injury site loosely adhered

2 31% to 70% of dura at injury site densely adhered

3 >70% of dura at injury site densely adhered

The slides were also evaluated for overall histological appearance (theoverall amount of fibrosis including new tissue, the density of thefibrosis, the vascularity of the new tissue filling in the defect site,and the presence or absence of foreign body response) The density of theoverall fibrosis and the level of the foreign body reaction wereestimated. The sections were scored using the following system:

0 No reaction seen

1 Mild reaction

2 Moderate reaction

3 Severe reaction

RESULTS

As seen in FIG. 12, the particle gel reduced epidural fibrosis (Table 2,p=0.0171). The particle gel samples had large amounts of materialdetected histologically within the laminectomy site. In the majority ofsections (11 of 18), there was no foreign body reaction to the material(Score=0). In the remaining sections, the foreign body reaction was mild(Score=1).

TABLE 2 Effectiveness Scores Number of Sections Percentage of SitesTreatment Score with Given Score with Given Score Control 0 10 19.6 1 1121.6 2 18 35.3 3 12 23.5 Particle Gel 0 9 50.0 1 6 33.3 2 2 11.1 3 1 5.6

Numerous modifications and alternative embodiments of the presentinvention will be apparent to those skilled in the art in view of theforegoing description. Accordingly, this description is to be construedas illustrative only and is for the purpose of teaching those skilled inthe art the best mode for carrying out the present invention. Details ofthe structure can vary substantially without departing from the spiritof the invention, and exclusive use of all modifications that comewithin the scope of the appended claims is reserved. It is intended thatthe present invention be limited only to the extent required by theappended claims and the applicable rules of law.

All literature and similar material cited in this application,including, patents, patent applications, articles, books, treatises,dissertations and web pages, regardless of the format of such literatureand similar materials, are expressly incorporated by reference in theirentirety. In the event that one or more of the incorporated literatureand similar materials differs from or contradicts this application,including defined terms, term usage, described techniques, or the like,this application controls.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way.

While the present inventions have been described in conjunction withvarious embodiments and examples, it is not intended that the presentteachings be limited to such embodiments or examples. On the contrary,the present inventions encompass various alternatives, modifications,and equivalents, as will be appreciated by those of skill in the art.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail can be made without departing fromthe scope of the appended claims. Therefore, all embodiments that comewithin the scope and spirit of the following claims and equivalentsthereto are claimed.

The invention claimed is:
 1. A medical device, comprising: a medicaldevice structure; and a coating formed on at least a portion of themedical device structure; wherein the coating comprises fatty acidparticles made from a method of forming fatty acid particles thatcomprises the steps of: (a) associating a cross-linked, fattyacid-derived biomaterial with a cryogenic liquid; and (b) fragmentingthe bio material/cryogenic liquid composition, such that fatty acidparticles are formed, wherein the fatty acid particles comprise fattyacids cross-linked directly to each other by bonds formed during acuring process, wherein the source of the fatty acids is a fish oil andwherein the particles have a distribution of size of about 1-20 μm(v,0.1), 21-40 μm (v,0.5), and 41-150 μm (v,0.9).
 2. The medical deviceof claim 1, wherein the medical device structure is a vascular graft,hernia mesh, thin film, or stent.
 3. Therapeutic fatty acid particlescomprising fatty acids cross-linked directly to each other by bondsformed during a curing process, wherein the source of the fatty acids isa fish oil and wherein the particles have a distribution of size ofabout 1-20 μm (v,0.1), 21-40 μm (v,0.5), and 41-150 μm (v,0.9).
 4. Theparticles of claim 3, further comprising a therapeutic agent.
 5. Acomposition comprising the particles of claim 3 further combined with afish oil.
 6. A therapeutic film comprising pressed therapeuticparticles, wherein the therapeutic particles are therapeutic fatty acidparticles according to claim
 3. 7. The therapeutic film of claim 6,wherein the therapeutic particles further comprise a therapeutic agent.8. A method of treating an adhesion in a subject in need thereof,comprising administering to the subject the therapeutic fatty acidparticles of claim
 3. 9. The method of claim 8, wherein the fatty acidparticles further comprise a therapeutic agent.
 10. The method of claim8, wherein the fatty acid particles are pressed into a film.
 11. Theparticles of claim 3, wherein the mean particle size of the particles isin the range of about 1 micron to about 50 microns.